Long-term sphere culture cannot maintain a high ratio of cancer stem cells: a mathematical model and experiment.
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ABSTRACT:
SUBMITTER: Peng T
PROVIDER: S-EPMC3217918 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Umbilical cord matrix stem cells (UCMSC) have generated great interest in various therapeutic approaches, including liver regeneration. This article aims to analyze the specific characteristics and the potential occurrence of premalignant alterations of UCMSC during long-term expansion, which are important issues for clinical applications. UCMSC were isolated from the umbilical cord of 14 full-term newborns and expanded in vitro until senescence. We examined the long-term growth potential, senescence characteristics, immunophenotype and multilineage differentiation capacity of these cells. In addition, their genetic stability was assessed through karyotyping, telomerase maintenance mechanisms and analysis of expression and functionality of cell cycle regulation genes. The tumorigenic potential was also studied in immunocompromised mice. In vitro, UCMSC reached up to 33.7 ± 2.1 cumulative population doublings before entering replicative senescence. Their immunophenotype and differentiation potential, notably into hepatocyte-like cells, remained stable over time. Cytogenetic analyses did not reveal any chromosomal abnormality and the expression of oncogenes was not induced. Telomere maintenance mechanisms were not activated. Just as UCMSC lacked transformed features in vitro, they could not give rise to tumors in vivo. UCMSC could be expanded in long-term cultures while maintaining stable genetic features and endodermal differentiation potential. UCMSC therefore represent safe candidates for liver regenerative medicine.
| S-EPMC3739759 | biostudies-other
Project description:We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device. This approach demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double-layered bead composed by an inner layer of alginate and an outer layer of Puramatrix. We also demonstrated the maintenance and expansion of Multiple Myeloma cells in this construction. Here, the presented microfluidic technique is applied to construct a 3D biomimetic model to recapitulate the human hematopoietic stem cell niche using double-layered hydrogel beads cultured in 10% FBS culture medium. In this model, the long-term maintenance of the number of cells and expansion of hHSCS encapsulated in the proposed structures was observed. Additionally, a phenotypic characterization of the human hematopoietic stem cells generated in the presented biomimetic model was performed in order to assess their long-term stemness maintenance. Results indicate that the ex vivo cultured human CD34+ cells from bone marrow were viable, maintained, and expanded over a time span of eight weeks. This novel long-term stem cell culture methodology could represent a novel breakthrough to improve Hematopoietic Progenitor cell Transplant (HPT) as well as a novel tool for further study of the biochemical and biophysical factors influencing stem cell behavior. This technology opens a myriad of new applications as a universal stem cell niche model potentially able to expand other types of cells.
| S-EPMC7830102 | biostudies-literature
Project description:BACKGROUND:Cancer stem cells (CSCs) constitute 1-2 % of cancer tissue and are a major cause of tumor metastasis and recurrence. The culture environment is important in maintaining CSCs in vitro. Sphere formation is one method of culturing CSCs. In this study, we identified and validated optimal culture conditions for sphere formation in hepatocellular carcinoma cells. METHODS:Huh7 and HepG2 cells were plated in three different media types and were allowed to form spheres. To confirm the pluripotency of sphere cells, the expression of stem cell markers was evaluated. EpCAM, Connexin 32, and Connexin 43 expression was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). The expression of E-cadherin, ?-catenin, CD90, and CD133 was evaluated using immunocytochemistry. Flow cytometry was performed to confirm the presence of stem cell markers CD133 and Connexin 32. RESULTS:Cells maintained in medium containing growth factors ((DMEM(+))GF) showed greater cell proliferation than cells in media with fetal bovine serum (FBS) (DMEM(+)FBS) or without FBS (DMEM(-)FBS). Cells cultured in DMEM(+)FBS medium exhibited greater proliferation after 20 days in culture than cells cultured under the other two conditions. Cells cultured in DMEM(-)FBS medium did not proliferate; therefore, this condition was removed from further analysis. RT-PCR and flow cytometry showed that sphere-forming cells cultured in DMEM(+)GF and DMEM(+)FBS media had similar expression of stem cell markers. CONCLUSION:Therefore, growth factor-free medium is an adaptable, efficient, and cost-effective tool for in vitro cultivation of CSCs.
| S-EPMC4599746 | biostudies-literature
Project description:Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this study was to evaluate the ability of three dimensional floating spheres to preserve embryological and biological properties of this sub-population. In addition, the expression of the inwardly rectifying potassium channel Kir4.1, Fas and FasL was investigated in 3D-sphere derived hDPSCs. Our data showed that 3D sphere-derived hDPSCs maintained their fibroblast-like morphology, preserved stemness markers expression and proliferative capability. The expression of neural crest markers and Kir4.1 was observed in undifferentiated hDPSCs, furthermore this culture system also preserved hDPSCs differentiation potential. The expression of Fas and FasL was observed in undifferentiated hDPSCs derived from sphere culture and, noteworthy, FasL was maintained even after the neurogenic commitment was reached, with a significantly higher expression compared to osteogenic and myogenic commitments. These data demonstrate that 3D sphere culture provides a favorable micro-environment for neural crest-derived hDPSCs to preserve their biological properties.
| S-EPMC5985438 | biostudies-other
Project description:Grapevines are exposed to gradual drought and salinity
2010-08-09 | PRD000224 | Pride
Project description:The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomy-treated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.
| S-EPMC5387414 | biostudies-literature
Project description:Three?dimensional (3D) cultures are indispensable for capturing tumor heterogeneity in colorectal cancer (CRC) in vitro. Although 3D cultures (such as sphere?forming assay and organoid culture) can partially preserve the morphological and molecular characteristics of primary CRC, whether these 3D cultures maintain the long?term stemness of cancer stem cells (CSCs) remains largely unknown. In the present study, spheres and organoids were generated side by side using individual primary CRC specimens, then respectively processed as serial passages. The results revealed that during serial passages, the percentage of CSCs (such as cluster of differentiation?133+ and Wnt+ cells) in organoids and the tumor?initiating capacity of organoid?derived cells were constant, while they gradually increased in the sphere?derived cells. Furthermore, during serial passages, resistance to chemotherapeutic agents (including 5?fluorouracil and oxaliplatin) in sphere? and organoid?derived cells was evaluated. The results indicated that the percentage of chemoresistant cells was constant in serial organoid cultures; however, it gradually increased in the serial sphere?forming assays. Taken together, the results of the present study comprehensively demonstrate that, with regard to long?term culture in vitro, organoid culture may be useful in maintaining tumor heterogeneity and the levels of chemoresistant cells, while the sphere formation assay enriches for CSCs and chemoresistant cells.
| S-EPMC6365025 | biostudies-literature
Project description:The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hopx+ colitis-associated regenerative stem cell (CARSC) population that functionally contributes to mucosal repair in mouse models of colitis. Hopx+ CARSCs, enriched for fetal-like markers, transiently arose from hypertrophic crypts known to facilitate regeneration. Importantly, we established a long-term, self-organizing two-dimensional (2D) epithelial monolayer system to model the regenerative properties and responses of Hopx+ CARSCs. This system can reenact the "homeostasis-injury-regeneration" cycles of epithelial alterations that occur in vivo. Using this system, we found that hypoxia and endoplasmic reticulum stress, insults commonly present in inflammatory bowel diseases, mediated the cyclic switch of cellular status in this process.
| S-EPMC6904908 | biostudies-literature
Project description:Sustainability of maize-based cropping systems is a major challenge for southern Africa, yet the demand for maize as staple food and animal feed in the region continues to increase. A study was conducted on a sandy clay loam (220 g clay kg-1 soil) at Domboshawa in Zimbabwe to investigate the long-term effects of organic resource quality and application rate, and nitrogen (N) fertilization on soil chemical properties and maize (Zea mays L.) productivity. Crotalaria juncea (high quality), Calliandra calothyrsus (medium quality), cattle manure (variable quality), maize stover and Pinus patula sawdust (both low quality) were incorporated into soil at 4.0 t C ha-1 (high rate) and 1.2 t C ha-1 (low rate) at the start of each cropping season for nine consecutive years. At both high and low application rates, each of the five organic resources was applied in combination with or without mineral nitrogen (N) fertilizer at 120 kg N ha-1. The nine-year period saw maize grain yields declining by 22% to 84% across treatments despite increases in soil organic carbon, total N and available P from 6% to 80%. Crotalaria, Calliandra and manure led to a less steep yield decline. Exchangeable calcium (Ca), magnesium (Mg) and potassium (K), and soil pH explained much of the variation in yield patterns observed under the different organic resource applications. Maize grain yield was positively correlated with exchangeable Ca (r = 0.51), Mg (r = 0.62) and K (r = 0.53), and soil pH (r = 0.49), but negatively correlated with other soil properties over the 9-year period. We concluded that declining soil exchangeable basic cations were the underlying causes of decreasing maize productivity, and was aggravated by use of low rates of organic resource inputs, particularly with N fertilization. Current nutrient management and fertilizer recommendations that emphasize inorganic N, P and K significantly undervalue the role played by organic resources in sustainability of maize cropping systems in southern Africa.
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Project description:Recently we and two other groups have shown that human spermatogonial stem cells (SSCs) have the potential to become pluripotent in vitro in defined culture conditions and to differentiate into cells of the three embryonic germ layers. This discovery could open new avenues for autologous cell-based therapy in degenerative diseases, bypassing the ethical and immunological problems related to the human embryonic stem cells. In addition, human SSCs could be used to treat infertility in cancer survival children. However, in order to reprogram SSCs into pluripotency, or to preserve them for repopulation of infertile testes, the first and limiting step is to have access to a highly purified human SSC population that could be multiplied and efficiently cultured in vitro maintaining their molecular and cellular characteristics. Although various studies have attempted to identify molecular markers of human SSCs, to date there is still limited information related to the specific markers that could be used for their isolation and optimized purification that allows long-term in vitro culture of isolated human SSCs. Here using SSEA-4 as an optimal marker for isolation of a subpopulation of SSCs, we show that SSEA-4 positive cells express the highest level of SSC genes compared to other subpopulations isolated with different markers, and can be maintained in culture for over 14 passages which we were unable to obtain with other SSCs markers including GPR125 and ITGA6. In addition, we have established a new technology for cell sorting and long-term culture of human SSC-SSEA-4 positive cells that maximizes the purity and viability of the sorted cells. Our findings are crucial and could be used for the most efficient isolation, purification and long-term culture of SSCs for clinical applications in regenerative medicine, or for preparation of human SSCs for autologous treatment of infertility in cancer survival children.
| S-EPMC3898576 | biostudies-literature